As chromatographers we face a bewildering number of choices when it comes to selecting the correct high performance liquid chromatography (HPLC) column for an analysis. True, we may be able to find a suggested stationary phase in the literature, but are the column dimensions applicable, will that column geometry work well with our system and allow us to attain our required detection levels? This installment of "The Essentials" establishes a number of "rules of thumb" that can be applied when selecting stationary-phase type and column dimensions to somewhat ease the burden that comes with such a proliferation of choice. 

Column length is typically predicated by the resolution required from a "traditional" HPLC system and in this sense we mean systems that have not been designed or adapted to minimize extracolumn volume or cannot achieve system back pressures above 400 bar. High resolution is typically required when separating samples with many components or when many chemically similar compounds are being separated. Beware that HPLC is a diffusion-limited technique and that above analyte retention factor (

) values of around 10, increasing retention will have little or no effect on resolution because of decreases in efficiency caused by an increase in longitudinal molecular diffusion. So you may need to adjust the eluotropic strength of the eluent to reduce overall retention time. 

Column internal diameter is typically chosen depending on analytical requirements and system limitations. It dictates the speed of analysis and influences method sensitivity. Use 4.6-mm i.d. columns when working with traditional HPLC systems described above, or 3-mm i.d. columns if efficiency loss due to extracolumn volume is not too great. Use 2.1-mm i.d. columns when you are using low-extracolumn-volume systems to save solvent, increase sensitivity (such as when working with a limited sample), or to reduce flow rate while still working at a reasonable eluent linear velocity (when working with electrospray ionization mass spectrometry [ESI-MS] systems, for example). 

The particle size of the stationary phase support affects the efficiency of a separation and one typically needs high efficiency when trying to separate a few components in a short time or many components in a reasonable time. Smaller particle sizes bring higher efficiency; however, this is usually at the expense of an increase in system back pressure, unless flow rate is adjusted downward. The most widely used particles are 5 µm in diameter and provide very reasonable efficiencies when used with traditional HPLC systems. Other particles with diameters such as 3.0 µm or 3.5 µm provide higher resolution and shorter analysis times, but beware of increases in system back pressure and also note that as particle size reduces so does the retaining frit porosity so greater care must be taken with sample and eluent preparation. Sub-2.0-µm particles are typically used with ultrahigh-pressure liquid chromatography (UHPLC) systems, which are capable of dealing with high system back pressures (1000 bar and greater) and can generate very high efficiencies for high resolution or very fast separations. We now also have a choice in particle morphology with the availability of core–shell particles, which give rise to high efficiencies (approaching those of sub-2-µm particles) with much lower system back pressures. The lower back pressure provides the advantage of allowing their use on traditional HPLC systems. 

Pore size describes the average pore diameter of the pores on the surface of the silica packing material. Pores of 6–12 nm are used with analytes below ~2000 Da and 30 nm pores are used for analytes above 4000 Da. For the 2000–4000 Da range you should experiment to see which pore size gives the best retention and efficiency. Wider-pore columns tend to provide a higher surface area and therefore are more "loadable" — that is, more analyte mass can be applied before peak shape deterioration (asymmetry) becomes a problem. 

			Type II (B) silicas are less acidic than Type I (A) and therefore give rise to less peak tailing when analyzing polar or ionizable analytes (especially basic molecules).	

Select hydrophobic stationary phases (C18, C8) when differences in analyte hydrophobicity are large and can be exploited to affect a separation. Typically, this is when the carbon to heteroatom ratio within the analyte is large. Hydrophobic stationary phase ligands do not work well in 100% aqueous eluents and are not well suited for the retention of more polar analytes. 

Modified alkyl phases (those with more polar functional groups embedded within the ligand) are used when separating analytes with different (polar) functional groups and where exploiting differences in the bulk hydrophobic properties of analytes does not produce an effective separation. Typically, these phases can be used with 100% aqueous eluent systems and are better at retaining polar analytes. 

Phenyl-containing phases interact strongly with analytes containing p electron systems (aromatic, unsaturated) and will retain these analytes in preference to those with no p electrons.   

Cyano and fluorinated phases interact strongly with basic, nitrogen-containing, and halogenated species and should be used when the analytes contain these species. 

Amino, diol, and silica phases are traditionally used for the separation of polar analytes in both reversed-phase and normal-phase modes. Lately they have also found utility in hydrophilic interaction liquid chromatography (HILIC) mode in which highly organic eluent systems are used to retain polar analytes using polar stationary phases. 

Articles

The Essentials

Some "rules of thumb" can be applied to selecting stationary-phase type and column dimensions.

HPLC Column Selection

This article presents a new method to evaluate the temperature shift observed in analytical temperature rising elution fractionation (ATREF). The evaluation is based on the dependence of the measured peak temperature as a function of heating rates. Application of the proposed method does not require any knowledge of the fluid circuit characteristics geometry and avoids the use of narrow preparative TREF standards. The results are found to be more accurate than the method that is usually applied.

Since the publication of the Wild article (1) in 1982, analytical temperature rising elution fractionation (ATREF) has been a method of choice for polyolefin characterization. In 1991 Exxon introduced the first patents in which polyethylenes were described by their compositional distribution breadth index (CDBI), an ATREF parameter equivalent to the polydispersity index of the molecular weight distribution. Exxon was followed by other material producers like Dow and Cryovac, and the number of patents incorporating claims covered by ATREF analysis steadily increased to about 30 patents per year. At the beginning, the ATREF systems were built by modifying gel permeation chromatography (GPC) systems. The increasing demand for ATREF measurements fostered the introduction of the first automated commercial ATREF system by Polymer Char in 1996.

No standardized method was published for this technique, and most of the patents make reference to Wild's publication (1). 

To calculate the CDBI, several standards of polyethylene with narrow compositions and known short-chain branching characteristics are injected into the ATREF unit and chromatograms are recorded as a function of time while monitoring the column temperature. These data are used to construct thermograms, in which the detector signal is plotted as a function of the column temperature. The recorded column temperature is usually higher than the real melting temperature of the eluted fraction because of the delay between melting in the column and detection. This delay depends on several experimental parameters, including column dimensions, tubing characteristics between the column and detector, and heating and elution rates. These equipment-specific parameters prevent the use of any short chain branches (SCB)-elution temperature calibration curves available in the literature (1–3). Actually, as recommended by the commercial ATREF manufacturer (4), the use of an identical procedure and identical equipment is mandatory to be able to compare samples. 

The procedure would be greatly simplified with a robust method to calculate the temperature shift, allowing for the correlation between results obtained with different instruments and methods. 

To circumvent that problem, the temperature shift can be calculated based on the tubing volume between the column and the detector. As the heating rate and the flow rate during the heating step are usually constants, the tubing volume can be converted into temperature shift using the following relationship: 

				temperature shift (°C) = heating rate (°C/min) × tubing volume (mL)/flow rate (mL/min) [1]			

As this method assumes a detailed knowledge of the tubing and does not take into consideration the heat transfer delay in the column heating system, it is expected that the calculated temperature shift will be lower than the actual one. 

An alternative method to evaluate the temperature shift is to analyze narrow preparative TREF standards (5,6). In this case, the temperature shift is given by the difference between the measured ATREF peak temperature and the preparative TREF temperature of the standard; the difficulty of the method is the fact that narrow TREF standards, isothermally eluted in a temperature interval of about 3 °C, are not easily available. Additionally, the high preparative column volume introduces measurement errors close to the temperature interval used to elute the preparative fraction (3 °C). Another potential issue with this method is the fact that usually the analytical TREF solvent (trichlorobenzene) is different from the solvent used in preparative TREF (xylene). 

In this article, we present a new method to evaluate the temperature shift for ATREF that does not require either the detailed knowledge of the tubing between the column and the detector or the availability of narrow standards obtained by preparative TREF. To apply the method, it is possible to use any unimodal sample such as a commercial metallocene polyethylene or high-density polyethylene. The method is based on the idea that the temperature shift is zero for isothermal elution steps. Because finite heating rates are used with ATREF, we propose to find the real melting temperature by analyzing the same sample with different heating rates and to extrapolate the measured elution temperatures to isothermal conditions. After finding the real melting temperature by extrapolation, we can then use a general rule to calculate the temperature shift for a given heating rate. 

The analyses were done using a TREF-Crystaf model 300 instrument (Polymer Char). The TREF unit is integrated into an Agilent Technologies model 7890 GC system oven, which is kept as originally supplied by the manufacturer with the exception that the main switch also activates the TREF dedicated power supply and all other electronics. TREF control software is used to set the oven temperature program through one of the computer serial ports. The elution step was performed with an Intelligent Pump Series 300 (IP300, Flom Corporation), located close to the oven and controlled by the TREF software. The concentrations of the eluted polymer fractions were measured with the built-in infrared detector, using wavelengths between 2800 and 3000 cm

Four commercial metallocene polyethylenes were analyzed using the ATREF system. The selected samples have narrow short-chain branching distributions and the following densities: 0.923, 0.934, 0.947, and 0.955 g/cm

Each sample was dissolved in 1,2,4-trichlorobenzene (TCB Spectropure dry, Biosolve Chimie, CAS 120-82-1) at 150 °C to obtain a 2 mg/mL solution. The solvent was stabilized with 0.3% of antioxidant (butylated hydroxytoluene, BHT, CAS 128-37-0). About 300 µL of the hot solution was injected at 150 °C in the ATREF column (100 mm × 0.25 in.) filled with metallic shots provided by Polymer Char. The column filled with solution was fast cooled (1 °C/min) to 100 °C, and then was further cooled to 30 °C with a cooling rate of 0.1 °C/min. After the cooling step, the polymer was eluted from the column, using a flow rate of 0.5 mL/min. Before starting the heating to 130 °C, the column was maintained at 30 °C for 30 min. For each sample, four different heating rates were used: 0.2, 0.5, 1.0, and 2.0 °C/min. 

Figure 1 is an overlay of the four elution profiles (trefograms) recorded for the 0.947-g/cm

  density polyethylene. The total elution step increases from 80 min for a heating rate of 2 °C/min to 530 min for a heating rate of 0.2 °C/min. Trefograms of the other three standards showed similar behavior. 

Figure 1: Overlaid chromatograms (infrared detector signal as a function of time) recorded using different heating rates for the 0.947-g/cm3 polyethylene.			

Figure 2 shows the same elution profiles as a function of the recorded temperature during the elution step. The overlay of the four thermograms is representative of the results. One can note that the peak temperature and the peak width increase with the heating rate. 

Figure 2: Overlaid thermograms (infrared detector signal as a function of recorded temperature) for the 0.947-g/cm3 polyethylene.			

The measured temperatures for each experiment were automatically corrected by the commercial TREF software based on the tubing lines between the TREF column and detector cell, and the elution rate. The results are given in Table I. The differences between the peak temperatures obtained with 0.2 and 2.0 °C/min are about 3 °C. 

Table I: Automatically corrected temperatures for all experiments, calculated by TREF software based on the tubing lines between the TREF column and detector cell			

In Figure 3 the measured peak temperatures are plotted as a function of the heating rates for the different polyethylenes used in this work. The extrapolated elution temperatures at zero heating rates represent the real melting temperatures. The obtained parallel lines of the linear dependence of the elution temperatures vs. heating rates for the different densities implies the existence of a unique parameter to calculate the temperature shifts. For a flow rate of 0.5 mL/min, the observed average slope of 5.1 min corresponds to the time needed for a polymer fraction to be melted and travel to the detector. This time delay takes into account the tubing volume as well as other parameters such as the heat transfer between the oven and the column, and the necessary time to solubilize the melted polymer. 

Figure 3: Plot of measured peak temperatures as a function of heating rates for four metallocene polyethylenes.			

With this parameter, the temperature shift can be calculated for any heating rate with the following relationship: 

temperature shift (°C) = heating rate (°C/min) × time delay (min)  [2]    			

With this equation, the measured temperatures for the 16 ATREF experiments were corrected; the results are presented in Table II. A maximum difference of 0.4 °C was noted, which is about 10% of the error recorded with the previous method. This difference is close to the variability of the ATREF method when determining the peak temperature by repeated measurements (for six measurements using a heating rate of 1 °C/min done on HDPE NIST 1475, the average uncorrected elution temperature is 101.3 °C, and the standard deviation is 0.2 °C). 

Table II: Corrected temperatures for all samples, calculated using the average slope of the linear dependence between the measured elution temperatures vs. heating rates 			

A new and simple method is proposed that allows for evaluating the temperature shift in analytical temperature rising elution fractionation (ATREF). This technique is based on the variation of the measured peak temperature as a function of the heating rate during the elution step and does not require the detailed knowledge of the instrument tubing or the availability of standards obtained by preparative TREF. To apply the method, it is possible to use any polyolefin sample, as long as it has a narrow composition distribution. 

Apart from measuring the correct melting temperature with higher precision, the method opens the possibility of creating "universal" calibration curves (SCB vs. temperature) for each type of comonomer. Such a procedure would improve the interlaboratory reproducibility for the calculation of CDBI and other parameters characterizing the chemical composition distribution of different types of polyethylenes. 

The method was initially developed on a commercial ATREF system, but should be valid with in-house ATREF instruments with different configurations. 

L. Wild, T.R. Ryle, D.C. Knobeloch, and I.R. Peat, 

F. Chen, R.A. Shanks, and G. Amarasinghe, 

Polymer Char TREF User Manual, page 2–17 ( 2011).			

A.G. Boborodea, D. Daoust, A. Jonas, and C. Bailly, 

US 7389678, A.G. Boborodea, C. Bailly, D. Daoust, A. Jonas, and B. Nysten, Column for analytical temperature rising elution fractionation (ATREF). 

are with Certech ASBL in Seneffe, Belgium. Direct correspondence to: 

Peer-Reviewed Articles

A new method is presented to evaluate the temperature shift observed in analytical temperature rising elution fractionation (ATREF).

Evaluating the Temperature Shift in Analytical Temperature Rising Elution Fractionation

 Awards, and the winners continue to honor the legacy of chromatographers before them. The 2013 Lifetime Achievement in Chromatography Award is granted to Peter W. Carr, and the 2013 Emerging Leader in Chromatography Award is presented to Davy Guillarme. A complete listing of our prestigious award winners over the years is shown in Tables I and II.  												

Summarizing or even touching on the vast accomplishments of a person like Peter W. Carr, whose career spans more than 50 years, is a daunting task, but let's start at the beginning. Carr majored in chemistry at the Polytechnic Institute of Brooklyn where he worked with Professor Louis Meites and graduated in 1965. During his undergraduate years, Carr actively pursued his career in the summer months. From 1961 to 1963 he was a laboratory technician at the American Molasses Company, and in 1965 he was a research assistant at Brookhaven National Laboratory, moving up to research associate in 1966. From there, he went on to get his PhD in chemistry under Professor Joseph Jordan at Pennsylvania State University followed by postdoctoral studies at Stanford University Medical School. 

Fellow Penn State graduate Richard A. Henry, formerly with Keystone Scientific and Penn State University, explained that Carr started his research in the field of electrochemistry and moved to calorimetry to prepare a thesis. His interest in separations didn't develop until after he received his PhD and started to pursue an academic career. Henry says we are all blessed that Carr chose to focus on separations. "The fields of electrochemistry and calorimetry did not need Pete Carr, but separations did," he said.  

 Table I: Winners of the LCGC Lifetime Achievement in Chromatography Award

Following his postdoctoral associate work at Stanford from 1968 to 1969, Carr accepted an assistant professor position in the chemistry department at the University of Georgia, where he was promoted to associate professor in 1975. In 1977, Carr moved to the University of Minnesota (UMN) as an associate professor in the department of chemistry. In 1981, he was made a full professor at UMN and he remains there to this day. During his tenure there, which spans more than three decades, Carr has held the titles Associate Director of the Cooperative Research Center for Bioanalytical Processing (1987–1990) and Associate Member with the Graduate Faculty for Microbial Engineering (1990–present).  

Table II: Winners of the LCGC Emerging Leader in Chromatography Award

Carr is well known for his pioneering contributions to the fundamental theory and practice of liquid chromatography (LC), with more than 400 highly cited papers and 18 patents under his belt. His early work focused on understanding retention and selectivity in reversed-phase LC, high performance liquid chromatography (HPLC), and gas chromatography (GC), including the development of various linear solvation energy relationships and the "hydrophobic subtraction model" of reversed-phase selectivity that is now in wide use. This work served as the inspiration for Ilja Siepmann, Carr's colleague at UMN, and Mark Schure to undertake more detailed computational modeling of retention in HPLC. 

Figure 1: A collage of various group members, friends, and events from Carr’s career. 			

When we reached out to many of Carr's colleagues, past students, and friends — a group in which those labels are interchangeable — the resounding response to our question about Carr's biggest contributions to the field was, "Where does one start?" William E. Barber, an R&D manager for chemistries at Agilent Technologies and one of Carr's former PhD students, put it nicely: "Pete has contributed to and solved a wide variety of problems in separation science. He has been a critical contributor in the advancement of our understanding of the fundamental theory of HPLC, LC media, and stationary-phase development, and most recently, the areas of high-speed HPLC and two-dimensional [2D] LC." 

Figure 2: Carr, an avid fisherman, is shown here landing a fish.			

Victoria McGuffin, a chemistry professor at Michigan State University, shared Barber's feelings."One of Carr's strengths is that you can ask 10 separation scientists about his greatest contribution and get 10 different responses — all equally valid," she said. McGuffin cited Carr's work with detailed thermodynamic measurements and solvatochromic modeling of retention and selectivity among his most significant accomplishments. "These studies, together with their careful and thorough interpretation, have provided a clearer understanding of the role of the mobile and stationary phases in liquid chromatography as well as the relative contributions of polarizability and dipolarity, hydrogen-bond donor, and hydrogen-bond acceptor interactions," she said. 

Figure 3: Carr taking in the beautiful scenery of St. Paul, Minnesota, during a fishing trip on the Mississippi River.			

 Lifetime Achievement Award winner and a retiree from LC Resources, has known Carr since the early 1980s and coauthored some 20 papers with him. Snyder cited the importance of Carr's work with columns for use at extremes of pH and temperature, along with the development and commercialization of zirconia as an ultrastable support for HPLC, followed by hypercrosslinked and other phases to match. "These columns permitted the use of temperatures greater than 100 °C for improving the speed of analysis in HPLC, including application to reversed-phase, ion-exchange, and protein separations," said Snyder. "Pete is also the inventor of tandem thermally tuned HPLC, a really clever idea that allows a continuous variation of column selectivity during method development." 

Dwight Stoll, an assistant professor at Gustavus Adolphus College, former graduate student of Carr's, and the 2011 winner of the 

 Emerging Leader in Chromatography award, echoed Snyder's comments regarding Carr's work with zirconia. "Pete's work was important for the development of zirconia-based stationary phases for HPLC," Stoll said. "Since his work on this in the 1990s and 2000s, several other groups have picked up the work and several materials have been commercialized." One such commercialization was ZirChrom Separations, Inc., a company that Carr cofounded in 1995 with Clayton McNeff and Steven Rupp. Carr also served as the first president of the company.  

Carr's most recent groundbreaking work is in ultrafast and 2D LC. "Pete's prior experience in both the thermodynamics and kinetics of HPLC has had a major impact on 2D-HPLC for the analyses of highly complex samples," said Snyder. "Two-dimensional separation is very demanding because it requires large values of 

in the first dimension and fast separation (for example, <10 s) in the second dimension," he said. "The technology he has developed is also applicable to high-speed gradient elution." 

Figure 4: A gathering prior to a recent MCF symposium. Standing (from left): Larry Potts, Don Hagen, Dick Henry, John Dolan, Pete Carr, and Bill Barber. Seated (from left): are Larry Bowers and Peter Johnson.			

Barber feels this work is the highlight of Carr's career. "Pete seriously considered retiring several years ago but came to realize that the 2D LC project was in many ways the capstone of his career contributions in that it employed and integrated several of the contributions he had made earlier in his career, that is, theory, high-speed LC, and high stability media and stationary phases," he said. 

Carr arguably has one of the world's leading and most active groups working in 2D separations, Stoll added. 

Contributions to the Professional Community

Carr's contributions go beyond his own research, as he has been a very active member of the professional community. For instance, Carr cofounded the Minnesota Chromatography Forum (MCF) with Steve Anderson, Larry Bell, Larry Bowers, and Don Hagen in 1978 and became the first president of the group. "The MCF was born in the minds of a few Minnesota area chromatographers in early 1978," the organization's web site says (1). "It took final shape on St. Patrick's Day in 1978 over a pitcher of green beer. In 1979, a constitution was written and passed by the members, declaring the objectives of the MCF to be . . . to maintain and promote for education, discussion and exchange of information with respect to all fields of chromatography."   

"All those present on St. Patrick's Day felt that there was a great need for education in chromatography that was not being met," said Larry Bowers of the United States Anti-Doping Agency and one of Carr's earliest PhD students. Bowers explained that the idea was to have quarterly meetings and an annual symposium at which chromatographers could learn from the experts and present their work on a local level. "The response from the community was overwhelming," Bowers said. The forum is active today, with three evening sessions held each year with invited speakers ranging from local experts to leading international chromatographers and an annual three-day spring symposium. The spring symposium continues to be a big success, bringing in about 250 attendees and 100 exhibitors annually. 			

In addition, Carr was chair of the Chromatography and Separations Chemistry Subdivision of the American Chemical Society's Division of Analytical Chemistry from 1987 to 1989 and the program chair of the International Symposium on Column Liquid Chromatography in 1994. Also, he has been an organizer of and presenter at many chromatographic sessions at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) and the International Symposium on High-Performance Liquid-Phase Separations. Carr has also served on the editorial advisory boards of 

Analytical Chemistry, Talanta, Microchemical Journal, Journal of Chromatography, Chromatographia, Separation Science, 

Carr's willingness to collaborate with other scientists also should not be overlooked. Bowers explained that Carr has an amazing ability to see the big picture and his education at an engineering school and work with Meites gave him the tools to integrate many disciplines into theoretical modeling. "He is very willing to share his knowledge and the credit for any product that arises," said Bowers. 			

As one might expect, someone with this many achievements is sure to receive accolades from the scientific community. Carr has received a long list of honors throughout his career. Some of his most notable awards are the L.S. Palmer Award from the Minnesota Chromatography Forum (1984); the Merit Award from the Chicago Chromatography Discussion Group (1987); the Benedetti-Pichler Award from the American Microchemical Society (1990); the Award in Analytical Chemistry from the Eastern Analytical Symposium (1993); the Dal Nogare Award of the Delaware Valley Chromatography Forum at Pittcon (1996); the ACS Award in Analytical Chemistry (2009); the A.J.P. Martin Gold Medal of the Chromatography Society (2010); and the Csaba Horvath Award of the Connecticut Separation Science Council and the Hungarian Chromatography Society (2010) — just to name a few. 

Carr has also been the recipient of many honors for his teaching, including the Award for Outstanding Contributions to Postbaccalaureate, Graduate, and Professional Education from the University of Minnesota Alumni Association (2002). In the same year, he was entered into the Academy of Distinguished Teachers at UMN. 

As his teaching awards indicate, Carr has given the same level of dedication and intellect evidenced in his research to the numerous students he has taught. So far, Pete has mentored 14 MS students, 50 PhD students, and 36 postdoctoral associates. The results, notes McGuffin, are impressive. She explained that some of Carr's students have chosen academic careers at high-quality undergraduate institutions and leading research universities all around the word, while others have gone on to prominent industrial careers at small companies or even Fortune 500 companies. "The quality and breadth of his student's careers are a tribute to Professor Carr's dedication as a teacher and mentor," she concluded. 

His students agree. "I think the aspect of Peter's professional work that is the most under-recognized is his extraordinary commitment to the personal and professional development of his graduate students," said Stoll. "Over the years, he has been incredibly loyal to them, supporting them well beyond their years in graduate school." 

Stoll went on to explain that Carr spends a great deal of time providing advice to his former students on employment and professional decisions, often connecting them to one another professionally or introducing them to key people in the chromatography field. Stoll has taken Carr's mentorship to heart and practices his methods with his own students at Gustavus Adolphus College. "In this way, Peter's commitment to this aspect of his teaching has far reaching consequences beyond just the immediate generation of graduate students," he said. 

Barber, another former student, mirrored Stoll's statements about Carr's teaching style. "The thing that impressed me the most about Pete as a thesis advisor was his dedication to his students," he said. "I always felt that he was not only willing to spend as much time as needed coaching his students but that he relished this time." Barber also discussed Carr's knack for knowing when to "let go" of his students, as some became independent researchers quickly, while others needed more coaching. "However, as soon as a student was ready to make the project his or her own, Pete was ready to let the student run with it," Barber said. 

Barber also pointed out that Carr always asks his PhD students to investigate both a theoretical and practical aspect of their work. "The value of this, of course, is that it allows the student to contribute to the fundamental understanding of the science as well as producing something that is practically useful to others in the field," he said. 

As Carr's students move on in their careers, he strives to maintain personal contact and friendships with them, as evidenced by the many stories shared with us for this article. Bowers is one example of that type of relationship: He was Carr's third PhD student and later became a long-time colleague at UMN.  

And just as Carr formed lasting relationships with his students, he maintained strong connections with those that preceded him. Carr was very proud of the analytical chemistry history at UMN and visited with I.M. Kolthoff while he was still alive. "Despite his theoretical strengths, Pete's favorite saying was from Kolthoff: 'Theory guides, experiment decides,'" said Bowers. The analytical chemistry division softball team was even named "I.M. Kolthoff and You're Not."  

In recent years, Carr became an avid fisherman and his love of the water was not lost on his students. Several photos included in this article are of Carr out fishing. Barber shared a story about picking Carr up on his 36-foot sailboat in the Chesapeake Bay just before the 2008 HPLC conference in Baltimore, Maryland. "Instead of driving, we sailed down the bay and up the Patapsco River into the Inner Harbor, where I docked the boat in a marina that was literally right next to the conference hotel," he said. "Pete got a real kick out of 'arriving' at the HPLC meeting under sail!" 

Stoll also shared some personal stories of fishing trips spent with Carr, explaining that in the cases where conferences are held near a good fishing destination (such as Pittcon in New Orleans, Louisiana) Carr would often take a day to test the waters before or after the conference. Carr also has favorite spots near his home, such as the Mississippi River and Lake Vermillion. "As a graduate student I came to understand that an invitation to go fishing with Peter was not only a time to enjoy the beauty of the Mississippi River valley, but also a time to talk about new ideas for chromatography research and recount some of the rich history of the chromatography community," said Stoll. "These times spent with Peter are some of the experiences I treasure most." 

's 2013 Emerging Leader in Chromatography winner, Davy Guillarme, received his Masters degree in science in 2001 and his PhD in analytical science in 2004, both from the University of Lyon in France. His PhD thesis was focused on an evaluation and the use of high-temperature liquid chromatography. He completed his postdoctoral work in 2005 at the University of Geneva in Switzerland, where he researched the application of capillary electrophoresis coupled to mass spectrometry (MS) for the analysis of biological matrices. From 2006 to 2010 he was an "assistant master" at the University of Geneva, University of Lausanne, and in 2010 he was promoted to senior lecturer, a distinguished position for someone so young.  

Guillarme's research covers a wide range of areas in analytical science, including fast and high-resolution liquid chromatography using state-of-the-art approaches such as ultrahigh-pressure liquid chromatography (UHPLC), core–shell technology, high mobile phase temperature, and the use of silica-based monoliths; chromatography analysis of intact biomolecules including therapeutic peptides, proteins, and monoclonal antibodies (mAbs); hydrophilic interaction liquid chromatography (HILIC) for the analysis of polar and other ionizable compounds; the evaluation of supercritical fluid chromatography (SFC) using conventional and sub-2-µm particles; and LC–MS and UHPLC–MS applied to pharmaceutical analysis.  

Guillarme's career has taken off at rocket speed, as his publications record attests. He has already published more than 77 peer-reviewed papers, and his recognition in the field (h-index = 23) is very high despite his young age.  

"This is a very creditable record considering that his first publication in a major scientific journal was as recently as 2004," noted Professor David McCalley, a professor of bioanalytical science at the University of the West of England. "This means that this large number of publications has been produced within the space of only eight years."  

Thus, Guillarme has been the author or coauthor of 10–12 publications per annum in the primary scientific literature during each of the past four years. In 2012, his work to date had received nearly 350 citations. "Two of these publications have well over 100 citations each, and thus can be classed as works of very significant impact," McCalley said.  

Some key contributions include his work in fast and high-resolution chromatography, particularly comparing those approaches using various types of samples. He was one of the first scientists to publish on the extensive possibilities offered by UHPLC instruments. He also created a free Excel calculator that can determine the isocratic and gradient conditions to be used when transferring a method between HPLC and UHPLC; this tool has been downloaded more than 7000 times (2). Guillarme has also co-edited a book, 

, with colleague Jean-Luc Veuthey. Recently, Guillarme published a series of papers that discuss the possibilities of various modern wide-pore reversed-phase LC phases for the analysis of large biomolecules. The results of this latest work demonstrate that reversed-phase LC can be a useful approach for the characterization of intact proteins and mAbs. 

Features

Profiles of Peter W. Carr, the Lifetime Achievement in Chromatography award winner, and Davy Guillarme, the Emerging Leader in Chromatography winner

The 2013 LCGC Awards

John Hinshaw's review of gas chromatography (GC) instruments and accessories that were newly on display at the Pittsburgh Conference in March 2013, or were introduced to the marketplace in the preceding year.

For the first time since its inception 64 years ago, the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) moved to Philadelphia, Pennsylvania, March 17–22, 2013. The newly expanded Pennsylvania Convention Center, which reopened in March 2011, was a good fit for Pittcon's exhibition with 1925 booths subscribed by 1011 exhibitors from 28 countries. Of these, 167 exhibitors displayed at Pittcon for the first time. Attendees seemed to find it easier to locate companies' booths, and the exhibitors did not seem to mind their generally smaller square footage as compared to previous years' shows. A number of companies often seen in the past at Pittcon declined to exhibit again this year, although their presence was felt in technical papers and posters, as well as in a soft gas chromatography (GC) system introduction in which the instrument was displayed in several of the company's channel partners' booths. 

The relocation to Philadelphia was a success with more than 18,000 registrants, over 40% of whom were first-time attendees. Registration was significantly higher than in the past two years. Of the attendees, 28% originated from outside the United States, which reflects the conference's continuing strong global presence. The conference organizers' emphasis on the technical program was evident this year with more than 2000 technical sessions presented in 78 symposia, 12 awards, 93 oral sessions, 12 workshops, and 62 posters. The short-course program also was very successful with more than 1400 participants in 100 courses. 

In 2014 Pittcon will return to Chicago, Illinois, and in 2015 the conference heads south to New Orleans, Louisiana. Other future locations include Atlanta, Georgia, and Orlando, Florida.  

This annual "GC Connections" installment reviews GC instrumentation and accessories shown at this year's Pittcon or introduced during the previous year. For a review of new GC and liquid chromatography (LC) columns and related accessories, please see the latest installments of "Column Watch" (1) in the April and May 2013 issues of 

The information presented here is based on manufacturers' replies to questionnaires, as well as on additional information from manufacturers' press releases, web sites, and product literature, and not upon actual use or experience of the author. During Pittcon, I took time to stroll around the convention aisles and see some of the new products firsthand as well as discover a number of items that weren't covered by the questionnaires. Every effort has been made to collect accurate information, but due to the preliminary nature of some of the material 

 cannot be responsible for errors or omissions. This article cannot be considered to be a complete record of all new GC products introduced this year at Pittcon or elsewhere because not all manufacturers chose to respond to the questionnaire, nor is all of the submitted information necessarily included here because of the limited space available and the editors' judgment as to its suitability.  

The year leading up to Pittcon 2013 saw continued strong interest in GC as demonstrated by the arrival of another significant crop of new GC instruments. An on-going resurgence of mass-spectrometry (MS) capable instrument systems plus new entries in miniaturized and portable devices underlined the importance of rapid and specific analytical results across all the market segments. From another direction, continuing concern over dwindling helium supplies has sustained general interest in hydrogen carrier gas, along with much discussion of alternate carrier gases including purified air. 

Agilent introduced the 7890B GC system, which features a number of incremental performance and ease-of-use improvements plus some new features related to mass-selective detection, inert sample path, and carrier-gas calculations. The instrument was seen in a number of the company's channel partners' booths, effectively accomplishing a soft product introduction for the new system. The 7890B GC system was accompanied by the introduction of an upgraded 5977A GC–mass-selective detector system, with a new source design and a new turbomolecular pump with a higher compression ratio that is designed for light gases such as hydrogen and helium. 			

Shimadzu introduced its Tracera GC system with a new barrier discharge ionization detector that can deliver up to 100 times greater sensitivity than a conventional thermal conductivity detection (TCD) system. Shimadzu also exhibited the GCMS-TQ8030 triple-quadrupole mass spectrometer with high scan speeds and the wide variety of ionization modes that such an instrument can support. And along with some new and updated samplers (see below), CDS Analytical added a GC–TCD system to its product line, for fixed-gas analysis of pyrolysates from its trapping pyrolyzer systems. 

Table III: Sampling systems, detectors, and accessories

Thermo Fisher Scientific introduced the TSQ 8000 Pesticide Analyzer system, which is a complete solution to facilitate multiresidue pesticide analysis. It is built around the company's TSQ 8000 triple-quadrupole GC–MS system, configured for pesticide analysis to help technician-level operators harness complex methods for GC–MS-MS analysis.

Table III: Sampling systems, detectors, and accessories (continued) 			

There were three new micro-GC products on display at Pittcon this year. From APIX, the GCAP portable micro-GC and Max-One compact hazardous-area GC systems are based on silicon micromachined valved inlets, capillary columns, and high-frequency nano-metric resonant detectors. The systems can use purified air or conventional compressed carrier gases. Existing applications cover alkanes, permanent gases, volatile organic compounds, and other materials. APIX shared the 2013 Pittcon Editors' Bronze Award. 			

Inficon introduced the Micro GC Fusion system with temperature programmable capillary column operation, which enables the analysis of C

+ hydrocarbons along with improved sensitivity. The system uses microelectromechanical system (MEMS) inlets and detectors with modular columns for simultaneous parallel separations, and features a touchscreen user interface and WiFi communications. 	

The GCX On-line Micro GC system from the Italian company Pollution is a rack-mountable modular multicolumn system for process and on-line applications. It features a MEMS-based high-sensitivity thermal conductivity detector, micro capillary columns, several injector modes, and accessories such as multistream samplers and a thermal trapping or desorption module. The system supports a number of high-speed applications such as natural gas and odorants, permanent gases, hydrocarbons, and solvents. 

Conference attendees were presented with a significant array of newly introduced or updated accessories for their GC systems, including some new detector developments and a number of items related to hydrogen carrier gas. Continued interest in managing volatile samples related to headspace, thermal desorption, or purge-and-trap dominated the sampling category by far.  

In the detector lineup, the AccuTOF GCv 4G time-of-flight mass spectrometer from JEOL USA, paired with the Zoex GC?GC interface and JEOL's unique electron ionization (EI)/field ionization (FI) ion source, combines comprehensive GC with high-resolution MS, and a choice of EI, chemical ionization (CI), and FI ion sources. 

And from DET, the Catalytic Combustion ionization detector provides selective detection of compounds containing chains of methylene functional groups such as petroleum hydrocarbons, fatty acid methyl esters (FAME), and triglycerides without response from aromatic or cyclohydrocarbons.  

The volatiles sampling category included the CDS-7400 Soil/Water purge-and-trap autosampler and model 7000E purge-and-trap system from CDS Analytical. The 72-position autosampler features three removable trays of 24 vials and automated water addition for soil samples, as well as internal standard addition. The 7000E purge-and-trap sampler is a smaller, more streamlined version of the company's model 7000 system and includes a newly updated computer interface in addition to the other features of its predecessor. 

Torion introduced a pair of related air-sampling products, the Clarion portable air samplers for use with either conventional traps or with Torion's needle trap devices, and the Fuzion portable sample concentrator. The sample concentrator accommodates up to three different modules, including sample desorption, heated headspace, purge-and-trap, and internal standard addition. 

From Shimadzu, the HS-20 headspace sampler series consists of two models: a loop model for traditional static headspace methods, and a trap model that adds dynamic headspace sampling for applications requiring greater sensitivity. The HS-20 sample tray holds up to 90 10-mL and 20-mL sample vials, and the oven can be heated to 300 °C for analysis of higher-boiling compounds. 

The TriPlus-300 headspace autosampler from Thermo Fisher Scientific is a valve-and-loop headspace autosampler with a 120-vial sample tray capacity and an 18-vial incubation oven overlap capacity with constant vial equilibrium intervals. The heated zones of the headspace unit can be set from 30 to 300 °C. When used with the company's Trace 1300 Series GC instruments, the TriPlus 300 headspace autosampler can be moved easily from one GC system to another without modifying the GC pneumatics, for confirmational purposes or to optimize workload. 

The TT24-7 Series 2 thermal desorber from Markes International is designed for near-real-time unattended monitoring of volatile and semivolatile organic compounds in air or gas. The desorber operates cryogen-free and unattended for extended periods and incorporates two electrically cooled traps operating in tandem. 

Frontier Laboratories' new Tandem micro-Reactor system provides process engineers and scientists a flexible approach for rapidly screening and evaluating catalysts. It has dual heated reactor zones mounted in tandem that interface with the split–splitless inlet of a GC–MS system. The reactor contains the catalyst under evaluation; catalytically transformed products are swept directly onto a tube or the instrument can be mounted on a benchtop GC–MS system for on-line analysis of transformed products: gases, liquids, or solids.   

From Gerstel, the Automated Filtration option for the company's MPS Multipurpose Sampler enables efficient automated cleanup of up to 98 samples or extracts combined with other sample preparation steps or with introduction to an LC–MS-MS or GC–MS system. Along with the sampler and related accessories, the filtration option is controlled and sequenced by Gerstel's Maestro software. 

A strong interest in hydrogen carrier gas continues this year, still spurred by the ongoing helium shortage. The lower cost and potentially higher separation speeds of hydrogen carrier are a plus for chromatographers, and the removal of carrier-gas and flame-gas tanks from the laboratory make the deployment of hydrogen generators an attractive alternative. Parker Balston introduced a new higher capacity hydrogen generator this year, the model H2PEMPD-1300 generator with 1.3-L/m capacity, a palladium purification membrane, and proton-exchange gas generation. F-DGS International exhibited several series of laboratory hydrogen generators with from 100–1200 mL/min capacities, including models for flame gas and carrier gas with options for trap regeneration, as well as models that combine air and hydrogen generation plus a hydrogen sensor for the GC oven or laboratory environment. 

The SilFlow microfluidic gas stream switching device from SGE is available in three configurations: three-port, four-port, and a five-port Deans switch. The devices consist of a chemically inert fluidic disk into which are etched microflow channels, with a cover plate that accommodates the company's SilTite finger-tight zero-dead-volume connectors. It can be used for capillary column and detector selection and for multidimensional GC configurations.  

I would like to thank the manufacturers and distributors that kindly furnished the requested information before, during, and after Pittcon 2013, which allowed a timely report on new product introductions. For those manufacturers who did not receive a preconference questionnaire this year and would like to receive one and be considered for early inclusion into Pittcon 2014 coverage, please send the name of the primary company contact, the mailing address, fax number, and e-mail address to Laura Bush, Editorial Director, 

, Advanstar Communications, 485F US Highway 1 South, Suite 100, Iselin, NJ 08830, Attn: 2014 New GC Products. Or email: 

 "GC Connections" editor John V. Hinshaw is a Senior Scientist at BPL Global, Ltd., in Hillsboro, Oregon, and a member of LCGC's editorial advisory board. Direct correspondence about this column to the author via e-mail: 

GC Connections

Gas chromatography instruments and accessories that were newly on display at Pittcon 2013

New Gas Chromatography Products for 2013

Gas Chromatography: Fundamentals, Troubleshooting, and Method Development

proed.acs.org/course-catalog/courses/GCPC/

GC Troubleshooting — Column & Detector Issues

www.chromacademy.com/chromacademy-essentials.html

Quick Guide to Capillary GC Stationary Phases

www.chromacademy.com/chromatography-GC-stationary-phases.html

www.chromacademy.com/practical-HPLC-workshop.html

Understanding HPLC Column Characterization and Selection

Core Shell Particles – Present and Future 

High Performance Liquid Chromatography Basics On Demand

proed.acs.org/course-catalog/courses/HWEB/

High Performance Liquid Chromatography: Fundamentals, Troubleshooting, and Method Development 

proed.acs.org/course-catalog/courses/HPTP/

www.restek.com/catalog/view/2010001/65908

Departments

Short Courses

This month's installment of "Column Watch" comprises Part II of our yearly report on new products introduced at Pittcon. Gas chromatography columns, sample preparation products, and hardware, accessories, and small tabletop instruments mainly for sample preparation are covered.

The Pittcon 2013 Conference and Expo (less formally known as the 64th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy) was held for the first time in the city of Philadelphia, Pennsylvania, on March 17–21, 2013. This year's event hosted over 1011 instrument manufacturers and laboratory suppliers in more than 1925 booths. In addition to attending the exposition, the conferees listened to more than 2200 technical presentations (orals, posters, workshops, invited and contributed talks, and award symposia), checked on numerous company seminar rooms, or attended one of 100 short courses. 

Although the attendance was slightly up this year compared to last year, the average attendance had been shrinking over the past several years. Still, Pittcon remains one of the most important international analytical exhibitions where companies introduce their latest instruments, instrument accessories, software, columns, and sample preparation and other consumable products. Several notable companies were missing again this year, but enough were present such that visitors had plenty of opportunities to investigate new (and old) technologies. 

The purpose of this report is to provide information about many of the new separation consumables and accessory products that were displayed at Pittcon 2013. In some cases, products that were introduced during 2012 but after Pittcon 2012 (1,2) may be included for reasons of completeness. The information is based on manufacturers' responses to a questionnaire mailed in early 2013. Because of space limitations and the fact that some manufacturers did not respond to the questionnaire, this report cannot be considered a comprehensive listing of all new products that were introduced in Philadelphia. However, over the years, these Pittcon introduction summaries have provided a good source of information that would be difficult for one individual to gather during the four days of the exhibition. In addition, the products introduced have shown definite correlations to current research, development, and application activity in the separation sciences. 

As in previous years, columns and other products recommended by their manufacturers primarily for biomolecule separations or sample preparation are denoted in the tables with the designation BIO. Some of these products may be used for general high performance liquid chromatography (HPLC) separations as well, but their main emphasis is for biological samples. 

In part I of this coverage (3), I described new column introductions in the areas of HPLC, reversed-phase LC, normal- and bonded-phase LC, hydrophilic interaction liquid chromatography (HILIC), supercritical fluid chromatography (SFC), ion-exchange and ion chromatography, size-exclusion chromatography (SEC), and specialty chromatography. In this installment, I will look at gas chromatography (GC) columns, sample preparation products, and hardware, accessories, and small tabletop instruments, mainly for sample preparation. 

 This year, I observed that there was a continuing trend in high-efficiency chromatography driven by dramatic increases in chromatographic efficiency, especially in HPLC or ultrahigh-pressure liquid chromatography (UHPLC). In addition, the introduction of more products with inert surfaces driven by instrumental increases in sensitivity especially through increasing use of tandem mass spectrometry (MS) techniques in both GC and LC. The development of systems with more inert flow paths including more PEEK-lined tubing, newer deactivated packing materials, deactivated surfaces such as fused-silica columns, vials, metal tubing, and GC injection liners are all being addressed to improve recovery of very low levels of analytes. 

In HPLC, there was a further increase for instrumental enhancements, hardware components, and accessories designed to work with UHPLC. Many application-specific columns for HPLC and GC that are tested with "real world" samples before shipment were noted. Again at Pittcon, a considerable number of new accessories, hardware, and devices were introduced that are designed to make the chromatographic laboratory more productive. 

 Overall, there were no real breakthroughs in HPLC column technology, but a refinement in particle sizes, stationary-phase technologies, and column dimensions was noted. Pittcon 2013 saw the introduction of a few more sub-2-µm columns for UHPLC, but not to the extent of several years ago. However, many new superficially porous particles (SPP, also known as core–shell particles), both particle sizes and phases, made their appearance including particles with diameters as small as 1.3 µm. UHPLC has now established itself as a viable solution to today's separation problems; at least nine major instrument vendors now address this market with instruments capable of operation at pressures as high as 19,000 psi. This year a few more HILIC columns appeared, but alternative reversed-phase selectivities, especially phenyl-hexyl and penta-fluorophenyl phases, were evident at the booths of a number of column companies. Still, in the number of introductions, reversed-phase HPLC maintained its dominance. Application-wise, chiral, protein (particularly monoclonal antibody columns), and ion chromatography or ion-exchange columns dominated. 

 GC column introductions were dominated by new stationary phases, especially with new levels of inertness. However, chromatographers in the food, environmental, and forensic areas are finding that there is more than column inertness that must be addressed. Thus, ultra-inert total flow systems are under development, driven by the increased sensitivity of mass spectrometers. Porous layer open tubular (PLOT) columns with built-in particle traps to capture any tiny solid phases flaking off the columns made their appearance. Specifically, chiral compounds, polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds (VOCs), semivolatiles, and fatty acid analyses were addressed in the applications' domain. A new polar ionic liquid column with much higher temperature capability than typical wax columns was shown for the first time. 

 Sample preparation products for automation, such as 96-well filtration and solid-phase extraction (SPE) plates and plate seals and SPE micropipette tips, seemed to be in vogue. Because of the popularity of protein-crash approaches for the analysis of drugs and metabolites in biological fluids, filters (96-well plates and single tubes) designed to remove both precipitated protein and lipids or phospholipids were introduced. Affinity SPE phases for proteins and aflatoxins as well as a family of mycotoxins removal cartridges were noted. QuEChERS (quick, easy, cheap, effective, rugged, and safe; pronounced "catchers") is still "catching on" as the favored sample preparation technique for pesticides in fruits and vegetables. Some interesting phase separation columns might bring renewed interest to liquid–liquid extraction (LLE). 

 More than 40 new hardware products, accessories, and benchtop instruments for LC, GC, and sample preparation were noted. This year I broke the hardware and accessories introductions from the benchtop instrumental introductions into two separate tables (see Tables III and IV). UHPLC-specific products (tubing, fittings, column hardware, and in-line filters) with higher pressure ratings and decreased dead volumes were introduced. Several automated sample preparation instruments contribute to minimize the labor-intensiveness and drudgery of manually preparing samples for chromatography were introduced by smaller instrument vendors. This year a number of workstations that do some (but not all) of the sample preparation steps were evident. Products for preparing solid materials for further workup included homogenizers, shakers, and solvent evaporation stations. A unique sample card for direct analysis in real time (DART) MS was introduced that did away with lengthy sample preparation steps. 

Although GC is considered a relatively mature separation technique, new GC columns continue to be introduced each year at Pittcon (Table I). This year saw the introduction of 11 new GC columns: nine wall-coated open tubular (WCOT) and two PLOT. These columns were introduced by seven different vendors.  

Applications-specific GC columns are always popular introductions at Pittcon. This year most of the column introductions were focused on specific applications. In the area of environmental, capillary columns for volatile organic compounds (as designated in the US EPA Methods 501, 601, and 624) and semivolatiles (EPA 525, 610, 625, 8100, and 8270D) were featured. As GC detectors, particularly mass spectrometers, become more sensitive, GC columns have had to keep up with the demand of more inertness but now users are demanding more inertness in other parts of the system from the injector to the detector, which puts pressure on the manufacturers to keep up with these market demands. So, now the inertness of the entire flow path is being questioned and liners, seals, tubing, fabricated parts, and so on are being deactivated by special treatment processes.  

Another application area that received attention was the analysis of polynuclear aromatic compounds in foods; two new columns were introduced that addressed this area. Polycyclic aromatic hydrocarbons (PAHs) are primarily formed by incomplete combustion or pyrolysis of organic matter and during various industrial processes. PAHs generally occur in complex mixtures which may consist of hundreds of compounds. Humans are exposed to PAHs by various pathways. While for non-smokers the major route of exposure is consumption of food, for smokers the contribution from smoking may be significant. Food can be contaminated from environmental sources, industrial food processing, and from certain home cooking practices. There are 15 PAHs that are on the European Food Safety Authority's (EFSA) list and 25 on the expanded US EPA list (Method 610), but many more potentially carcinogenic compounds may also contribute to human health. Since the larger fused ring PAHs are rather nonvolatile, higher temperatures are needed to elute them in a reasonable timeframe. In fact, in some cases, reversed-phase HPLC can do a better job but sometimes sensitivity issues arise (4). 

Chiral compounds are most often separated by HPLC, but for essential oils that are complex and volatile a new chiral column was introduced that allows enantiomeric separations without requiring undesirable derivatizations. Fatty acid methyl ester (FAMEs) analyses have been a mainstay for GC for years; a new capillary column that has improved temperature stability relative to the widely used free fatty acid phase (FFAP) stationary phase was displayed at Pittcon 2013. Figure 1 depicts the separation of FAMES in biodiesel. In recent years, biodiesel interest has really sparked as an alternative to petroleum-based fuels. The European standard method DIN EN 14103: 2011 is used to determine the ester content of FAMEs intended for use as pure biofuel or as a blending component for heating and diesel fuels. It also allows one to determine the linolenic acid methyl ester content to verify that the linolenic acid content is between 1% (m/m) and 15% (m/m). This method is suitable for FAMEs that contain methyl esters between C

Figure 1: Determination of FAMEs from biodiesel. Column: 30 m Ã 0.25 mm, 0.25-Âµm df Optima FFAPplus; carrier gas: helium; flow rate: 40 cm/s; oven temperature program: 70 Â°C for 1 min, then 70â240 Â°C at 30 Â°C/min; injection volume: 1 ÂµL, split 1:15; injection temperature: 260 Â°C; detection: MS with electron ionization; ion source temperature: 200 Â°C; interface temperature: 250 Â°C. Peaks (methyl esters): 1 = caproic acid (C6:0), 2 = caprylic acid (C8:0), 3 = capric acid (C10:0), 4 = lauric acid (C12:0), 5 = myristic acid (C14:0), 6 = palmitic acid (C16:0), 7 = palmitoleic acid (C16:1), 8 = stearic acid (C18:0), 9 = oleic acid (C18:1 cis), 10 = linoleic acid (C18:2 cis), 11 = nonadecanoic acid (C19:0), 12 = linolenic acid (C18:3), 13 = arachidis acid (C20:0), 14 = behenic acid (C22:0), 15 = erucic acid (C22:1 cis), 16 = lignoceric acid (C24:0), 17 = nervonic acid (C24:1 cis). (Courtesy of Machery-Nagel.) 

Two new PLOT columns were introduced, one by Restek and one by Agilent Technologies. PLOT columns are the GC columns of choice for the analysis of light gases and low boiling volatile compounds because of their highly retentive character. However, PLOT columns generally have the disadvantage that the stationary phase layer is not mechanically stable and can be damaged easily with stationary phase particle shedding as a result. PLOT stationary phase particles flowing to the detector result in signal spikes, which can interfere with analytical results. For multicolumn valved PLOT column applications, phase particles flowing downstream into valves, Deans switches, and column connectors can cause flow restriction and damaged valve components resulting in unplanned instrumental downtime and increased maintenance costs. A new PLOT column was introduced by Agilent that has built-in particle trapping capability at both ends of the column; one end to keep particles from reaching the detector while at the other end the particle trap keeps particles from being backflushed into switching valves. 

As indicated in Table II, Pittcon 2013 was another productive year with 24 new sample preparation products or families of products displayed. Not counted were sample preparation accessories (Table III) or sample preparation benchtop instruments (Table IV), which will be covered later. The most popular application areas included drugs and drug metabolites in biological fluids, pesticides and toxins in food, and isolation of tiny amounts of proteins and peptides. Formats included cartridges, 96-well plates, and disks.

Table II: Sample preparation products (continued) 

Many of the products were for SPE and just about every mode (reversed phase, strong and weak anion and cation exchange, affinity, adsorption and normal phase, partition, and mixed mode) was represented. A wide variety of media were introduced at Pittcon including polymer-based, graphitized carbon black, silica-based bonded phases, bare silica, alumina, titania, and diatomaceous earth.  

Table III: Chromatography and sample preparation accessories 

This year, sample preparation introductions were dominated by the 96-well plate format; obviously these products are destined for those interested in the application of automation. Very few chemists would have the patience to manually work with such tiny wells performing SPE, filtration, and other liquid handling operations. If one doesn't have a robotic system, there are manual handheld eight-channel pipettors that relieve some of the tedium; there are even systems that are semiautomated that have 96 individual pipettors (see Table IV). The 96-well plates were introduced for SPE, filtration, and liquid handling. After the 96-well operation takes place, samples are usually collected in a 96-well collection plate and presented to an autosampler or liquid handling robot. Samples left in the collection plate sometimes sit in the autosampler for hours before analysis. Newer solvent-resistant sealing mats have been developed so that contained liquids don't evaporate and autosampler needles don't bind to. A special gas-permeable microplate seal was introduced that would allow air or CO

An active area for which both single tubes and 96-well plates were introduced was the filtration devices for protein "crashing" experiments. Rather than using SPE and spending time developing methods, the trend in recent years has been to precipitate (crash) proteins out of biological fluids (for example, plasma) by adding an organic solvent like acetonitrile. The proteins can be removed by centrifugation (somewhat time consuming) or can be filtered through a membrane filter and the supernatant analyzed by LC–MS-MS for the drugs and other compounds. The optimum methodology is to first place the crash solvent (such as acetonitrile) in the tube or well, then add the plasma and agitate. However, with old style 96-well plates, the acetonitrile would start to drip through the bottom frit even before the plasma was added. Newer membranes have been designed that allow the organic solvent to be placed in the crash device and flow starts only when a vacuum is applied. Unfortunately, the protein crash supernatant liquid still contains a number of compounds that could interfere with MS detection mainly by causing ion suppression. Several new products were introduced that during filtration also remove the lipids and phospholipids that are some of the main culprits for ion suppression. 

Table IV: Sample preparation instrumentation 

Besides the normal SPE phases, newly introduced products included affinity phases that provided an activated linking group that could immobilize antibodies for protein capture or already containing immobilized antibodies that could selectively isolate aflatoxins or mycotoxins from a food or feed extract.  

Table IV: Sample preparation instrumentation (continued)

Additional products for QuEChERS were introduced at Pittcon 2013. This method is an emerging technique that is becoming increasingly popular in the area of multiresidue pesticide analysis in food and agricultural products. In QuEChERS, food and agricultural samples are first extracted with an aqueous miscible solvent (such as acetonitrile) in the presence of high amounts of salts (for example, sodium chloride and magnesium sulfate) or buffering agents (such as citrate) to induce liquid phase separation and stabilize acid and base labile pesticides, respectively. Upon shaking and centrifugation, an aliquot of the organic phase is subjected to further clean up using SPE. Unlike traditional methods using SPE tubes, dispersive SPE (d-SPE) cleanup is conducted by mixing bulk amounts of SPE sorbent and additional magnesium sulfate with the extract. After sample clean up, the mixture is centrifuged and the resulting supernatant can either be analyzed directly or can be subjected to additional minor treatment before analysis. Some new QuEChERS products were displayed that provided convenient features like easy-pour salt packets and flat-bottom centrifuge tubes. A new carbonized-silica or -alumina packing has been developed that doesn't have some of the undesirable properties of graphitized carbon black, allows elution of planar pesticides without toluene, and removes matrix or pigments from colored extracts. 

LLE is still a widely used sample preparation technique. The technique is often performed in separatory funnels and requires large amounts of solvent and lots of manual labor. This year's Pittcon saw the introduction of two phase separator products that are designed to make LLE easier. The SiliaPrep (Silicycle) and the Universal Phase Separator (Biotage) products are small tube-like devices that are designed to perform LLE. The Silicycle device will perform extractions with solvents heavier than water and the Biotage device will perform extractions with solvents heavier or lighter than water. Both work on the principle of a hydrophobic frit interface between the organic solvent and the aqueous solvent. The two solvents are actually in separate chambers or channels. The Silicycle SiliaPrep product has separate devices that handle 12–150 mL volumes and the Biotage device has a capacity of 70 mL. Both depend on gravity to move the fluids through the cartridge. Figure 2 provides a simple diagram of the Universal Phase Separator device. 

Figure 2: Principle of operation of the Universal Phase Separator device. See Table II for details. (Courtesy of Biotage.) 

Chromatography and Sample Preparation Accessories

New hardware products and accessories designed to make the life of the chromatographer easier are always popular introductions at Pittcon. This year I have separated the columns and sample preparation accessories (Table III) and small, benchtop dedicated instruments that are designated to automate sample preparation and other chromatographic techniques (Table IV). I will cover each area individually. 

Several products that can be useful for HPLC, UHPLC, and GC (plus MS and MS-MS) were shown this year. Analytical standards and reagents are always needed in a chromatography laboratory; most users gravitate to chemical suppliers or laboratory supply houses who can furnish the products that they need. Waters saw the opportunity and this year began production of chemicals needed for supporting chromatography instruments. Because the consumables (parts and supplies) business for chromatography represents a substantial market, last year Agilent Technologies launched the initial phase of its Crosslab products for competitors' gas chromatography equipment. This year, Agilent launched the next phase by making a wide range of LC consumables such as detector lamps, fittings, vials, and autosampler syringes available for its competitors' HPLC and UHPLC instruments. 

Vials are used by the millions in chromatography laboratories all over the world. The 2-mL vial has been the standard for decades, but the opening on a typical vial is quite small. J.G. Finneran, a glass company, introduced the Versa Vial, which has a 9-mm-wide opening to accommodate standard pipettes. Thomson Instruments displayed their version of a filter vial. A filter vial takes the place of a syringe filter, syringes, glass vial, and cap in a single unit. The vial has a plunger that presses on a movable membrane filter frit and forces the liquid to be filtered through the membrane leaving behind the particulates. Because the unit stays intact, it can be placed directly in an autosampler. 

HPLC, UHPLC, LC–MS, and Ion Chromatography Products

The advent of UHPLC has spurred a number of ultrahigh-pressure products. This year, additional high-pressure components that were introduced include fittings, PEEK-lined column hardware, PEEK-lined capillary tubing, and in-line filters. 

Buffers are always a pain to prepare. So, UCT has come up with an idea borrowed from QuEChERS salt packets and now supplies pre-weighed buffers in packets — just add water. So far, only two concentrations of phosphate and acetate buffers are being supplied. 

When performing trace analysis, analyte loss and recovery is of the utmost importance. In fact, any part of the GC flowstream that touches the sample (for example, inlets including liners, capillary columns, connecting tubings, and weldments) must be thoroughly deactivated. Part of this "new" requirement is the greatly increased level of detection sensitivity now available with today's mass spectrometers that serve as the most popular GC detectors. Since detectors now operate at the femtogram level, the tiniest amounts of analyte must get through the chromatograph to be detected. Thus, ultrainert systems will be required henceforth. 

Table IV shows 27 new products, mostly for sample preparation. Sample preparation still represents a major bottleneck in the laboratory because it is often a manual operation. Small benchtop instruments that automate some of the manual sample preparation steps can save time, eliminate tedium, and reduce errors. In this section, I cover benchtop instruments that range from robotic designs that perform multiple sample preparation procedures on small sample volumes to a single task such as solvent evaporation or mixing.  

Automated liquid handling instruments are probably the more sophisticated of the benchtop units. Typically, these instruments go well beyond moving liquid samples from one container to another. For example, the CTC Analytics PAL RTC Fully Automated Prep and Load Systems, given the right accessory modules, can perform tasks such as liquid injection, headspace sampling, solid-phase microextraction (SPME), derivatization, LLE, and beyond. It has robotic tool change capabilities to widen the liquid handling up to five different liquid injection procedures. With sophisticated software, sample workflows can be managed and it even has built-in time management tools to increase throughput and avoid wasted time while a task is being performed. Of course one must pay for such sophistication, and if all of these capabilities are not required, users can consider units such as the LH96 Automated Workstation from Omni International that will perform fewer automated tasks but is still adequate for some laboratories.  

At the other end of the spectrum of benchtop instruments are those that are designed to perform only one task but do it extremely well. For example, for hard and brittle samples in large pieces (such as rocks) where more than a few grams is required to get a representative sample, disk mills and pulverizers easily accomplish tasks of obtaining finely divided solids that can be easily sampled and extracted using conventional techniques. Accomplishing this task by hand would be a long and difficult process. SPE vacuum manifolds are another convenient product that speed up the SPE multistep process by batching multiple samples at a time. Evaporators that greatly speed up the drying process after collection of large volumes of analyte effluent from SPE, pressurized fluid extractors, and microwave extraction were prevalent at Pittcon. The preparation of biological samples by cell disruption and homogenization equipment save a lot of time. 

A product that was impressive to me was the DART-OS with OpenSpot sample cards. DART is an atmospheric pressure ion source that instantaneously ionizes gases, liquids, and solids in open air under ambient conditions. It was among the first ambient ionization techniques not requiring sample preparation, so solid and liquid materials can be analyzed by MS in their native state. Ionization can take place directly on the sample surface, such as currency bills, tablets, bodily fluids (blood, saliva, and urine), glass, plant leaves, fruits and vegetables, and even clothing. Liquids are analyzed by dipping an object (such as a glass rod) into the liquid sample and then presenting it to the DART ion source. The OpenSpot sample cards are a convenient way to get the sample into the source and basically don't require any elaborate sample preparation; samples are merely placed on the card by depositing a liquid or rubbing a solid material onto a wire mesh portion of the card. The card is then inserted into a holder on the source, which aligns the sample in its correct position and that's it. Figure 3 depicts a card ready to be placed into the system. 

Column Watch

New Chromatography Columns and Accessories at Pittcon 2013 Part II

Ultrahigh-pressure liquid chromatography (UHPLC) systems are designed to take full advantage of sub-2-µm particles, which yield higher resolution and increased sensitivity. Because of increased back pressure, UHPLC systems employ high-performance pumps that deliver 15,000 psi (1000 bar) or more of pressure. Some systems solve the back-pressure issue by elevating temperatures (via column oven), which lowers the viscosity of the mobile phase and allows it to move faster through the column. UHPLC systems have been shown to provide analysis times of less than 1 min. 

 HPLC vs. UHPLC purchasing: 2011â2015.			

The introduction of the UHPLC technology has forever changed the dynamics of the liquid chromatography market. Even though UHPLC has its drawbacks, including the high initial systems price tag compared to standard high performance liquid chromatography (HPLC), the technology has been steadily replacing standard HPLC systems in laboratories across the world. It is to no surprise that in SDI's recently conducted survey of liquid chromatography users, the majority of end users indicated having a UHPLC system in their laboratory. However, for those who did not, the lack of laboratory budget was the biggest hurdle in obtaining one.  

To understand the purchasing patterns when it comes to the standard HPLC and UHPLC technologies, SDI asked the participants to indicate the types of liquid chromatography systems they have purchased in 2011 and 2012 and what they plan on purchasing from 2013 to 2015. According to the responses, standard HPLC system purchases have been declining and are expected to steadily decline from 2011 to 2015, respectively, while UHPLC systems are experiencing a higher purchasing rate for the same time frame. 

The foregoing data were extracted and adapted from SDi's recently published Tactical Sales and Marketing (TSM) report titled 

LCGC North America-05-01-2013

HPLC Column Selection

Evaluating the Temperature Shift in Analytical Temperature Rising Elution Fractionation

The 2013 LCGC Awards

New Gas Chromatography Products for 2013

Short Courses

New Chromatography Columns and Accessories at Pittcon 2013 Part II

Market Profile: UHPLC Versus Standard HPLC

Peaks of Interest

Gradient Elution, Part III: Surprises

Vol 31 No 5 LCGC North America May 2013 Regular Issue PDF